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Abstract [en]

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

In thesis

Zhu, Lin

KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.

2017 (English)Doctoral thesis, comprehensive summary (Other academic)

Abstract [en]

A large number of proteins interact with biological membranes, either integrated in the membrane (PepTSo2), embedded on a membrane surface (5-lipoxygenase) or encircling a cutout of lipid bilayer (apolipoprotein1 (apoA-I). They function as transporters, receptors or biocatalysts in cellular processes like inflammation or cholesterol transport which are touched upon here. Malfunction of specific membrane proteins are the cause for several diseases or disorders.

Knowledge of protein structure supports understanding of its mechanism of function. Here, transmission electron microscopy (TEM) was used for structure determination. To obtain structure information to high resolution for membrane proteins, normally surrounded by lipids, demands specific methods and materials for stabilization. Stabilized in detergent the structure of the bacterial transporter PepTSo2 was shown to form a tetramer even bound to substrate. However, with a protein based stabilizer, Salipro, the structure of PepTSo2 could be determined to high resolution.

High density lipoprotein (HDL) in blood plasma, involved in the removal of cholesterol from peripheral tissues, have a central role in cardiovascular function, metabolic syndrome and diabetes.

The HDL-particle is composed of two copies of ApoA1 and around hundred lipid molecules. From TEM data, for the first time the clearly discoidal shape could be shown by 3-dimendional reconstructions. These were used for modelling the ApoA1 protein dimer by a "biased fitting" procedure. The results indicate how ApoA1 folds around a lipid bilayer in a disc-shaped structure.

Modified HDL called nanodiscs were here used to show the Ca2+ dependent binding of 5-lipoxygenase on the nanodisc bilayer and thereby increased production of the inflammatory mediator leukotrieneA4. Dimerization of 5-lipoxygenase inactivates these functions.